The following is a brief description of publications concerning ribozymes, and in particular, hairpin ribozymes. none are admitted to be the prior art to the pending claims, and all are incorporated by reference herein.
Six basic varieties of naturally-occurring enzymatic RNAs are known presently. Each can catalyze the hydrolysis of RNA phosphodiester bonds in trans (and thus can cleave other RNA molecules) under physiological conditions. Table I summarizes some of the characteristics of these ribozymes. In general, enzymatic nucleic acids act by first binding to a target RNA. Such binding occurs through the target binding portion of enzymatic nucleic acid which is held in close proximity to an enzymatic portion of the molecule that acts to cleave the target RNA. Thus, the enzymatic nucleic acid first recognizes and then binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cut the target RNA. Strategic cleavage of such a target RNA will destroy its ability to direct synthesis of an encoded protein. After an enzymatic nucleic acid has bound and cleaved its RNA target, it is released from that RNA to search for another target and can repeatedly bind and cleave new targets.
The enzymatic nature of a ribozyme is advantageous over other technologies, such as antisense technology (where a nucleic acid molecule generally simply binds to a nucleic acid target to block its translation) since the concentration of ribozyme necessary to affect a therapeutic treatment is lower than that of an antisense oligonucleotide. This advantage reflects the ability to the ribozyme to act enzymatically. Thus, a single ribozyme molecule is able to cleave many molecules of target RNA. In addition, the ribozyme is a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding to the target RNA, but also on the mechanism of target RNA cleavage. Single mismatches, or base-substitutions, near the site of cleavage can completely eliminate catalytic activity of a ribozyme. Similar mismatches in antisense molecules do not prevent their action (Woolf et al., 1992 Proc. Natl. Acad. Sci. USA, 89, 7305-7309). Thus, the specificity of action of a ribozyme is greater than that of an antisense oligonucleotide binding the same RNA site.
Van Tol et al., 1991 (Virology 180, 23) describe a hairpin ribozyme structure able to circularize. Hisamatsu et al., 1993 (Nucleic Acids Symp. Ser. 29, 173) describe hairpin ribozymes having a long substrate binding site in helix 1. Berzal-Herranz et al., 1993 (EMBO J. 12,2567) describe essential nucleotides in the hairpin ribozyme. Hampel and Tritz, 1989 (Biochemistry 28, 4929) describe a hairpin ribozyme derived from the minus strand of tobacco ringspot virus satellite [(-) sTRSV] RNA. Haseloff and Gerlach 1989 (Gene 82, 43) describe sequences required for self-cleavage reactions catalyzed by the (-) sTRSV RNA. Feldstein et al., 1989 (Gene 82, 53) tested various models oi trans-cleaving motifs derived from (-) sTRSV RNAs. The hairpin ribozyme can be assembled in various combinations to catalyze a unimolecular, bimolecular or a trimolecular cleavage/ligation reaction (Berzal-Herranz et al., 1992, Genes & Develop. 6, 129; Chowrira and Burke, 1992 Nucleic Acids Res. 20, 2835; Komatsu et al., 1993 Nucleic Acids Res. 21, 185; Komatsu et al., 1994 J. Am. Chem. Soc, 116, 3692). Increasing the length of helix 1 and helix 4 regions do not affect the catalytic activity of the hairpin ribozyme (Hisamatsu et al., 1993 supra; Chowrira and Burke, 1992 supra; Anderson et aL, 1994 Nucleic Acids Res, 22, 1096). For a review of various ribozyme motifs, and hairpin ribozyme in particular, see Ahsen and Schroeder, 1993 Bioessays 15, 299; Cech, 1992 Curr. Opi. Struc. Bio. 2, 605; and Hampel et aL, 1993 Methods: A Companion to Methods in Enzymology 5, 37.
This invention concerns an improved ribozyme based on the hairpin motif described by Hampel and Fritz 1989 supra; Feldstein et al., 1989 supra; Hampel et aL, 1990 Nucleic Acid Rest 18, 299; and Hampel et al., EP 0360257.
Hairpin ribozyme substrate complex comprises of two intermolecular helices formed between the ribozyme and the target RNA (helix 1 and helix 2). Length of helix 1 can be varied substantially without effecting the catalytic activity of the ribozyme (Hisamatsu et al., 1993 supra). However, the length of helix 2 is reported to be sensitive to variation. The length of helix 2 is normally between 3 and 5 base-pairs long (Hampel & Tritz, 1989 supra; Feldstein et al., 1989 supra; Haseloff and Gerlach, 1989 supra; Hampel et al., 1990 supra; Feldstein et al., 1990 Proc. Natl. Acad. Sci. USA 87, 2623). Several reports suggest that mutations within this helix significantly inhibit ribozyme activity (Hampel et al., 1990 supra; Feldstein et al., 1990 supra; Chowrira & Burke, 1991 Biochemistry 30, 8518; Joseph et al., 1993 Genes & Develop. 7, 130). It is also believed in the art that the length of helix 2 should be between 3 and 5 bp (Hampel et al., 1988 EPO 360 257; Hampel et al., 1993 supra, Cech, 1992 supra; von Ahsen and Schroeder, 1993 supra; Hisamatsu et al., 1993 supra, Anderson et al., 1994 supra).